p38 mitogen-activated protein (MAP) kinase but not p44/p42 MAP kinase is involved in prostaglandin E1-induced vascular endothelial growth factor synthesis in osteoblasts

We investigated the mechanism underlying vascular endothelial growth factor (VEGF) synthesis stimulated by prostaglandin E1 (PGE1) in osteoblast-like MC3T3-E1 cells. PGE1 induced the phosphorylation of both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. SB203580, a specific inhibitor of p38 MAP kinase, inhibited the PGE1-stimulated VEGF synthesis as well as PGE1-induced phosphorylation of p38 MAP kinase. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, which reduced the PGE1-induced phosphorylation of p44/p42 MAP kinase, had little effect on the VEGF synthesis stimulated by PGE1. AH-6809, an antagonist of the subtypes of the PGE receptor, EP1 and EP2, or SC-19220, an antagonist of EP1 receptor, did not inhibit the PGE1-induced VEGF synthesis. H-89, an inhibitor of cAMP-dependent protein kinase, and SQ22536, an inhibitor of adenylate cyclase, reduced the VEGF synthesis induced by PGE1. Cholera toxin, an activator of G(s), and forskolin, an activator of adenylate cyclase, induced VEGF synthesis. SB203580 and PD169316, another specific inhibitor of p38 MAP kinase, reduced the cholera toxin-, forskolin- or 8bromo-cAMP-stimulated VEGF synthesis. However, PD98059 failed to affect the VEGF synthesis stimulated by cholera toxin, forskolin or 8-bromoadenosine-3',5'-cyclic monophosphate (8bromo-cAMP). SB203580 reduced the phosphorylation of p38 MAP kinase induced by forskolin or 8bromo-cAMP. These results strongly suggest that p44/p42 MAP kinase activation is not involved in the PGE1-stimulated VEGF synthesis in osteoblasts but that p38 MAP kinase activation is involved.     

Somatostatin inhibits Akt phosphorylation and cell cycle entry, but not p42/p44 mitogen-activated protein (MAP) kinase activation in normal and tumoral pancreatic acinar cells

Somatostatin, or its structural analog SMS 201-995 (SMS), is recognized to exert a growth-inhibitory action in rat pancreas, but the cellular mechanisms are not completely understood. This study was undertaken to evaluate the effect of SMS on p42/p44 MAP kinases and phosphatidylinositol 3-kinase activation and to analyze expression of some cell cycle regulatory proteins in relation to pancreatic acinar cell proliferation in vivo (rat pancreas), as well as in the well-established tumoral cell line AR4-2J. We herein report that: 1) SMS inhibits caerulein-induced pancreatic weight and DNA content and abolishes epidermal growth factor (EGF)-stimulated AR4-2J proliferation; 2) SMS only moderately reduces the stimulatory effect of caerulein on p42/p44 MAP kinase activities in pancreas and has no effect on EGF-stimulated MAP kinase activities in AR4-2J cells; 3) SMS repressed caerulein-induced Akt activity in normal pancreas; 4) SMS has a strong inhibitory action on cyclin E expression induced by caerulein in pancreas and EGF in AR4-2J cells and as expected, the resulting cyclin E-associated cyclin-dependent kinase (cdk)2 activity, as well as pRb phosphorylation, are blunted by SMS treatment in both models; and 5) SMS suppresses mitogen-induced p27(Kip1) down-regulation, as well as marginally induces p21(Cip) expression. Thus, our data suggest that somatostatin-induced growth arrest is mediated by inhibition of phosphatidylinositol 3-kinase pathway and by enhanced expression of p21(Cip) and p27(Kip1), leading to repression of pRb phosphorylation and cyclin E-cdk2 complex activity.     

A protein factor for ras p21-dependent activation of mitogen-activated protein (MAP) kinase through MAP kinase kinase.

To identify the direct target molecule of ras p21 in higher eukaryotes, we have recently developed the cell-free system in which ras p21 activates mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK). In this cell-free system, the guanosine 5'-[gamma-thio]triphosphate- bound form of Ki-ras p21, but not the GDP-bound form, activates endogenous Xenopus MAP kinase as well as recombinant ERK2 in the presence of the cytosol fraction of Xenopus oocytes. We separated two protein factors from the cytosol fraction of Xenopus oocytes by column chromatography: one was the inactive form of MAP kinase kinase and the other was a factor tentatively named ras p21-dependent ERK-kinase stimulator (REKS). The former and latter showed M(r) values of approximately 45,000 and 150,000-200,000, respectively, as estimated by gel filtration. Both factors were necessary for Ki-ras p21-dependent activation of MAP kinase/ERK2. These results indicate that an additional protein factor (REKS) is essential for Ki-ras p21 to activate MAP kinase through MAP kinase kinase.     

Insulin-like growth factor binding protein-3 in extracellular matrix stimulates adhesion of breast epithelial cells and activation of p44/42 mitogen-activated protein kinase

IGF-binding protein-3 (IGFBP-3) is a multifunctional protein that regulates the potent mitogenic and antiapoptotic effects of IGF-I and IGF-II and exerts bioactivity independent of modulating IGF receptor activation. Previous studies have shown that in solution, IGFBP-3 binds constituent proteins of the extracellular matrix (ECM) such as fibronectin and collagen and is present in ECM deposited by fibroblasts in vitro; however, binding of IGFBP-3 to matrix has not been characterized, nor has its function in this environment been investigated. In this study, we show that IGFBP-3 binds to ECM deposited by human breast epithelial and cancer cells and neonatal human fibroblasts. IGF-I and heparin blocked binding of IGFBP-3 to matrix when added with the binding protein but were unable to displace IGFBP-3 already bound to the matrix. IGF-I bound to matrix-immobilized IGFBP-3 with approximately 25-fold reduced affinity compared with IGFBP-3 in solution. Mutation of the C-terminal basic domain of IGFBP-3 (228KGRKR-->MDGEA) resulted in markedly reduced binding to matrix compared with wild-type IGFBP-3, whereas mutation of the adjacent consensus heparin-binding domain (220KKK-->HSR) had relatively little effect. In the presence of matrix-bound IGFBP-3, adhesion of breast epithelial cells was increased by approximately 25%, and activation of the signaling pathway intermediate p44/42 MAPK was enhanced greater than 3-fold. These results indicate a previously unrecognized and potentially important role for IGFBP-3 in the extracellular matrix.     

Involvement of p44/p42 MAP kinase in insulin-like growth factor-I-induced alkaline phosphatase activity in osteoblast-like-MC3T3-E1 cells

It has been shown that insulin-like growth factor-I (IGF-I) stimulates the activity of alkaline phosphatase, a marker of mature osteoblast phenotype, in osteoblasts. In the present study, we investigated the involvement of the mitogen-activated protein (MAP) kinase superfamily in the IGF-I-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. IGF-I-stimulated alkaline phosphatase activity dose dependently in the range between 1 nM and 0.1 microM. IGF-I induced the phosphorylation of p44/p42 MAP kinase and p38 MAP kinase but not stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). PD98059 and U0126, specific inhibitors of the upstream kinase that activates p44/p42 MAP kinase, significantly suppressed the IGF-I-induced alkaline phosphatase activity. On the contrary, SB203580 and PD169316, specific inhibitors of p38 MAP kinase, failed to affect the activity induced by IGF-I. Specific inhibitors for phosphatidylinositol 3-kinase (PI3K)/Akt pathway (LY294002 and wortmannin) also had no significant effect on IGF-I-induced p44/p42 MAP kinase phosphorylation. The phosphorylation of p44/p42 MAP kinase induced by IGF-I was reduced by U0126. These results strongly suggest that p44/p42 MAP kinase among the MAP kinase superfamily plays a role in the IGF-I-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells.     

ERK and p38 MAP kinase activation are components of opioid-induced delayed cardioprotection

Opioids have been previously shown to confer acute and delayed cardioprotection against a prolonged ischemic insult. We have extensively characterized the signal transduction pathway mediating acute cardioprotection and have suggested a role for extracellular signal regulated kinase (ERK) in this cardioprotection. Therefore, we attempted to determine a role for ERK and the stress activated MAP kinase, p38, in opioid-induced delayed cardioprotection by using selective inhibitors of these pathways. All rats were subjected to 30 min of ischemia and 2 h of reperfusion (I/R). Control animals, injected with saline 48 h prior to I/R, had an infarct size/area at risk (IS/AAR) of 61.6 ± 1.6. 48-h pretreatment with TAN-67 (30 mg/kg), a δ₁-opioid receptor agonist, maximally reduced IS/AAR (31.2 ± 6.5). The involvement of ERK was examined with PD 098059, a selective pharmacological antagonist which inhibits the upstream kinase, MEK-1, that phosphorylates and activates ERK. PD 098059 (0.3 mg/kg) did not alter IS/AAR when administered alone (60.7 ± 4.9). However, PD 098059 (0.3 mg/kg) administration 30 min prior to TAN-67 (30 mg/kg) completely abolished cardioprotection (61.0 ± 7.6). The selective p38 inhibitor, SB 203580 (1.0 mg/kg), had no effect on IS/AAR in the absence of TAN-67 (53.1 ± 2.3). Additionally, SB 203580 (1.0 mg/kg) when administered prior to TAN-67 (30 mg/kg) partially abolished cardioprotection (51.3 ± 6.4). These results suggest that both ERK and p38 are integral components of opioid-induced delayed cardioprotection and may act via parallel pathways. Received: 21 August 2000, Returned for revision: 13 September 2000, Revision received: 18 September 2000, Accepted: 20 September 2000     

Mitogen-activated protein (MAP) kinases are involved in interleukin-1 (IL-1)-induced IL-6 synthesis in osteoblasts: modulation not of p38 MAP kinase, but of p42/p44 MAP kinase by IL-1-activated protein kinase C.

We previously reported that interleukin-1alpha (IL-1alpha)-induced activation of protein kinase C (PKC) via phosphatidylcholine-specific phospholipase C (PC-PLC) limits IL-6 synthesis induced by IL-1alpha itself in osteoblast-like MC3T3-E1 cells. In the present study, we further investigated the mechanism behind IL-1alpha-induced IL-6 synthesis in MC3T3-E1 cells. IL-1alpha time-dependently stimulated the phosphorylation of both p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase. PD98059, a specific inhibitor of the upstream kinase that activates p42/p44 MAP kinase, inhibited the IL-1alpha-induced IL-6 synthesis as well as the phosphorylation of p42/p44 MAP kinase induced by IL-1alpha. SB203580, a specific inhibitor of p38 MAP kinase, also reduced both the phosphorylation of p38 MAP kinase and the IL-6 synthesis. 1-Oleoyl-2-acetylglycerol, an activator of PKC, suppressed the IL-1alpha-induced IL-6 synthesis. Calphostin C, a specific inhibitor of PKC, or D-609, a specific inhibitor of PC-PLC, significantly enhanced the IL-1alpha-induced phosphorylation of p42/p44 MAP kinase without affecting the phosphorylation of p38 MAP kinase. The phosphorylation of p42/p44 MAP kinase by IL-1alpha was markedly increased in PKC-down-regulated MC3T3-E1 cells. Neither 12-O-tetradecanoylphorbol-13-acetate, known to be an activator of PKC, nor 1-oleoyl-2-acetylglycerol affected the phosphorylation of p38 MAP kinase induced by IL-1alpha. These results strongly suggest that IL-1alpha-induced IL-6 synthesis is mediated via activations of both p42/p44 MAP kinase and p38 MAP kinase in osteoblasts, and that PKC activated by IL-1alpha itself negatively regulates IL-6 synthesis at a point upstream from p42/p44 MAP kinase.     

Mediation of basic fibroblast growth factor-induced lactotropic cell proliferation by Src-Ras-mitogen-activated protein kinase p44/42 signaling

Basic fibroblast growth factor (bFGF), which is secreted from folliculostellate cells in the anterior pituitary, is known to be involved in the communication between folliculostellate cells and lactotropes during estradiol-induced lactotropic cell proliferation. We studied the role of MAPK p44/42 in bFGF-regulated cell proliferation using enriched lactotropes and the lactotrope-derived PR1 cell line. In cell cultures, bFGF increased cell proliferation of PR1 cells and enriched lactotropes. In both of these cell populations, bFGF also increased phosphorylation of MAPK p44/42. U0126, an inhibitor of MAPK p44/42, blocked the bFGF-induced activation of MAPK p44/42 as well as the bFGF-induced cell proliferation of enriched lactotropes and PR1 cells. Treatment of PR1 cells with bFGF increased the activity of Ras p21, whereas overexpression of a dominant negative mutant of Ras p21 abrogated the bFGF-induced activation of MAPK p44/42 in these cells. Furthermore, the Src kinase inhibitor PP1 suppressed bFGF-induced activation of MAPK p44/42 in both enriched lactotropes and PR1 cells. The Src kinase inhibitor PP1 also reduced bFGF activation of Ras p21 and cell proliferation in PR1 cells. On the other hand, the bFGF-induced activation of MAPK p44/42 in enriched lactotropes and PR1 cells was not affected by protein kinase C inhibitors. These data suggest that bFGF induction of lactotropic cell proliferation is possibly mediated by activation of Src kinase, Ras p21, and MAPK p44/42.     

Distinct roles of p42/p44ERK and p38 MAPK in oxidant-induced AP-1 activation and cardiomyocyte hypertrophy

Cardiac hypertrophy is an adaptive response to a number of heart diseases including myocardial infarction. Although it can be compensatory at first, sustained hypertrophy is often a transition to heart failure. We have found that cardiomyocytes in culture can survive mild doses of H₂O₂ but develop hypertrophy involving activation of p70 S6 kinase 1 (p70S6K1). Here, the role of p42/p44^ERK and p38 MAPK in oxidant-induced hypertrophy is tested. H₂O₂-induced phosphorylation (activation) of p42/p44^ERK and p38 within 10 min of 200 μM H₂O₂ exposure. Although p42/p44^ERK remained highly phosphorylated from 60 to 120 min, the level of p38 phosphorylation reached highest at 60 min and started to decline at 90 min. Inhibiting ERKs with PD98059 attenuated H₂O₂-induced AP-1 activation but did not affect H₂O₂-induced p70S6k1 activation or cardiomyocyte enlargement as measured by increases in cell volume and protein content. In contrast, the p38 inhibitor SB202190 has not inhibitory effect on AP-1 activation but partially prevented H₂O₂ from inducing p70S6K1 activation and cell enlargement. These data suggest that while p42/p44^ERK participates in gene expression associated with hypertrophy, p38 may regulate cell size increase by p70S6K1 activation.     

Felodipine inhibits nuclear translocation of p42/44 mitogen-activated protein kinase and human smooth muscle cell growth.

OBJECTIVE: Smooth muscle cell (SMC) proliferation contributes to vascular structural changes in cardiovascular disease. Ca(2+) antagonists exert antiproliferative effects and may also be clinically beneficial in the patients. However, the underlying mechanisms of action remain elusive. Activation of mitogen-activated protein kinases (MAPK), in particular p42/44mapk plays a central role in cell proliferation. We hypothesise that Ca(2+) antagonists inhibit cell proliferation by interfering with the p42/44mapk pathway in human SMC. METHODS: SMC were cultured from human aorta. Cell proliferation was analysed by [3H]thymidine incorporation. Activation of p42/44mapk and the nuclear target protein Elk-1 was analysed by phosphorylation and p42/44mapk nuclear translocation by confocal microscope. RESULTS: PDGF-BB (10 ng/ml) stimulated [3H]thymidine incorporation, phosphorylated p42/44mapk, caused nuclear translocation of the enzymes and phosphorylated the nuclear target protein Elk-1. Felodipine (10(-7) to 10(-5) mol/l) inhibited [3H]thymidine incorporation to PDGF-BB, had no effect on p42/44mapk phosphorylation. However, p42/44mapk nuclear translocation and Elk-1 activation stimulated by PDGF-BB were prevented by the Ca(2+) antagonist. CONCLUSION: Activation of p42/44mapk, subsequent nuclear translocation and activation of Elk-1 are essentially associated with human SMC proliferation. The Ca(2+) antagonist felodipine prevents p42/44mapk nuclear translocation (but not its activation) associated with inhibition of human SMC growth.     

Blueberry opposes beta-amyloid peptide-induced microglial activation via inhibition of p44/42 mitogen-activation protein kinase

Alzheimer's Disease (AD) is the most common age-related dementia, with a current prevalence in excess of five million individuals in the United States. The aggregation of amyloid-beta (A beta) into fibrillar amyloid plaques is a key pathological event in the development of the disease. Microglial proinflammatory activation is widely known to cause neuronal and synaptic damage that correlates with cognitive impairment in AD. However, current pharmacological attempts at reducing neuroinflammation mediated via microglial activation have been largely negative in terms of slowing AD progression. Previously, we have shown that microglia express proinflammatory cytokines and a reduced capacity to phagocytose A beta in the context of CD40, A beta peptides and/or lipopolysaccharide (LPS) stimulation, a phenomenon that can be opposed by attenuation of p44/42 mitogen-activated protein kinase (MAPK) signaling. Other groups have found that blueberry (BB) extract both inhibits phosphorylation of this MAPK module and also improves cognitive deficits in AD model mice. Given these considerations and the lack of reduced A beta quantities in behaviorally improved BB-fed mice, we wished to determine whether BB supplementation would alter the microglial proinflammatory activation state in response to A beta. We found that BB significantly enhances microglial clearance of A beta, inhibits aggregation of A beta(1-42), and suppresses microglial activation, all via suppression of the p44/42 MAPK module. Thus, these data may explain the previously observed behavioral recovery in PSAPP mice and suggest a means by which dietary supplementation could mitigate an undesirable microglial response toward fibrillar A beta.     

Transforming growth factor-beta3 stimulates lactotrope cell growth by increasing basic fibroblast growth factor from folliculo-stellate cells

Recently, we have shown that transforming growth factor-beta3 (TGFbeta3) mediates estradiol's mitogenic action in primary cultures of mixed anterior pituitary cells. In some cell types, TGFbeta isoforms stimulate cell proliferation via a paracrine mechanism by increasing growth stimulatory peptide growth factors. Whether such a mechanism exists in pituitary cell culture was examined in the studies presented here. The data demonstrate that unlike the response of lactotropes in mixed pituitary cultures, cultures of enriched lactotropes, obtained by Percoll gradient separation, did not proliferate in response to TGFbeta3 treatment. The lactotropic cells of the RC-4B/C cell line, a cell line that contains all of the hormone-secreting cell types of the anterior pituitary but is devoid of folliculo-stellate (FS) cells, did not proliferate in response to TGFbeta3 unless RC-4B/C cells were cocultured with FS cells. Enriched lactotropes cocultured with FS cells also demonstrated a proliferative response to TGFbeta3. Media collected from FS cells treated with TGFbeta3 stimulated the proliferation of lactotropes in enriched cultures. TGFbeta3 increased the release of basic fibroblast growth factor from FS cells. Immunoneutralization of basic fibroblast growth factor in FS cell-conditioned medium inhibited the growth stimulatory action on lactotropes. These data provide evidence for a novel mechanism of TGFbeta3 action involving cell-to-cell interaction in the anterior pituitary between lactotropes and FS cells during estrogen-induced mitogenesis.     

高血压患者血清抗AT1-受体抗体对血管平滑肌细胞p44/p42丝裂原活化蛋白激酶表达的影响

目的已有研究表明抗血管紧张素Ⅱ受体1型(AT1-受体)自身抗体具有提高心肌细胞收缩频率的受体激动剂样作用,本研究观察该抗体对血管平滑肌细胞p44/p42丝裂原活化蛋白(MAP)激酶的影响,探讨高血压患者血清中抗AT1-受体自身抗体的生物学活性及其作用机制.方法我们从16例ELISA检测抗AT1-受体阳性的高血压患者血清中提取纯化了免疫球蛋白,用Western等方法检测抗体对培养大鼠血管平滑肌细胞的作用.结果发现抗AT1抗体可诱导平滑肌细胞p44/p42丝裂原活化蛋白(MAP)激酶的表达,与血管紧张素Ⅱ相似,并且这一效应可被AT1受体阻断剂氯沙坦抑制.结论P44/p42 MAP激酶是平滑肌细胞增殖肥厚的重要信号分子,抗AT1-受体抗体通过受体介导血管平滑肌细胞p44/p42 MAP激酶的表达,提示抗AT1-受体抗体具有引起血管平滑肌细胞肥厚增殖的作用.     

Inhibition of the p44/42 MAP kinase pathway protects hippocampal neurons in a cell-culture model of seizure activity

Excessive release of glutamate and the subsequent influx of calcium are associated with a number of neurological insults that result in neuronal death. The calcium-activated intracellular signaling pathways responsible for this excitotoxic injury are largely unknown. Here, we report that PD098059, a selective inhibitor of the calcium-activated p44/42 mitogen-activated protein kinase (MAP kinase) pathway, reduces neuronal death in a cell-culture model of seizure activity. Dissociated hippocampal neurons grown chronically in the presence of kynurenate, a broad spectrum glutamate-receptor antagonist, and elevated amounts of magnesium exhibit intense seizure-like activity after the removal of these blockers of excitatory synaptic transmission. A 30-min removal of the blockers produced extensive neuronal death within 24 h as assayed by the uptake of trypan blue and the release of lactate dehydrogenase. Phospho-p44/42 MAP kinase immunoreactivity after 30 min of seizure-like activity was present in many neuronal somata and dendrites as well as some synaptic terminals, consistent with both the presynaptic and postsynaptic effects of this pathway. The addition of PD098059 (40 μM; EC₅₀ = 10 μM) during a 30-min washout of synaptic blockers inhibited the phosphorylation of p44/42 MAP kinase and reduced both the trypan-blue staining (n = 13) and the release of lactate dehydrogenase (n = 16) by 73% ± 18% and 75% ± 19% (mean ± SD), respectively. The observed neuroprotection could be caused by an effect of PD098059 on seizure-like events or on downstream signaling pathways activated by the seizure-like events. Either possibility suggests a heretofore unknown function for the p44/42 MAP kinase pathway in neurons.     

Endothelin-mediated vascular growth requires p42/p44 mitogen-activated protein kinase and p70 S6 kinase cascades via transactivation of epidermal growth factor receptor.

Endothelin-1 (ET-1), a potent endothelium-derived vasoconstrictor peptide, exerts a growth-promoting effect on vascular smooth muscle cells, implicating its pathogenic role in vascular remodeling. To gain insight into the cellular and molecular mechanism whereby ET-1 induces vascular growth, we studied whether transactivation of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor, are required for activation of p42/p44 mitogen-activated protein (MAP) kinase and p70 S6 kinase (p70S6K), and subsequent growth-promotion by ET-1 in cultured rat vascular smooth muscle cells. Immunoblotting with antiphosphotyrosine antibody revealed that ET-1 rapidly (within 2 min) and transiently induced tyrosine phosphorylation of several proteins, among which 180-kDa protein was shown to be EGFR. ET-1 rapidly increased association of EGFR and Shc with glutathione-S-transferase-Grb2 fusion protein. The ET-1-induced activation of MAP kinase was reduced by an EGFR kinase inhibitor (AG1478) but not by a platelet-derived growth factor receptor kinase inhibitor (AG1296). AG1478 dose-dependently decreased ET-1-stimulated MAP kinase activity as well as [3H]leucine and [3H]thymidine uptake. The ET-1-induced tyrosine phosphorylation of EGFR, as well as MAP kinase activation, was inhibited by an ETA receptor antagonist and intracellular Ca2+ antagonists but not by an ETB receptor antagonist, pertussis toxin, or protein kinase C inhibitors. In addition, dominant negative mutant of H-Ras and a MAP kinase kinase (MEK-1) inhibitor (PD98059) completely blocked ET-1-induced MAP kinase activation as well as [3H]leucine and [3H]thymidine uptake. Both AG1478 and PD98059 inhibited ET-1-induced phosphorylation and activation of p70S6K. Furthermore, rapamycin, a selective inhibitor of mammalian target of rapamycin, completely blocked ET-1-stimulated [3H]leucine and [3H]thymidine uptake. These results suggest that ETA receptor-mediated vascular growth by ET-1 requires both MAP kinase and p70S6K cascades mediated partly via Ca2+-dependent EGFR transactivation.